System and method for microwave cell lysing of small samples

ABSTRACT

Efficient cell lysis in small samples, i.e., samples less than one milliliter, is achieved by exposing the sample to microwave radiation in the frequency range of 18 to 26 GHz. The sample containing cells is supported in a wave-guide cavity, and a microwave source provides microwave radiation to the input port of the wave-guide cavity. A computer controls the frequency and source power level of the microwave radiation produced by the microwave source. The computer also monitors the input power level of the microwave radiation at the input port by means of an input power measuring instrument, the output power level at the output port by means of an output power measuring instrument, and the temperature of the sample by means of a thermocouple. In this way, the computer can control the operating parameters to achieve efficient cell lysis.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of microwave technology. Moreparticularly, this invention relates to a system and method for heatingsmall samples and for lysing small samples of cells.

2. Description of Related Art

Cell lysis is the process of breaking apart the cell membrane to releasethe cell contents. In many cases, the cell contents of interest are thenucleic acids, i.e., the DNA or RNA. For example, cell lysis istypically performed on cells to release the DNA or RNA as the first stepin amplification processes such as PCR.

Currently, cell lysing is most commonly accomplished chemically, such asby using detergents, solvents, or enzymes. However, this approach hasthe disadvantage of requiring a supply of the appropriate chemicals,with the associated storage and disposal problems.

Cell lysing can also be accomplished thermally. For example, the samplecan be placed in thermal contact with a thermal block, such as a hotplate. However, such conventional heating techniques often take arelatively long time, which can result in excessive evaporation of thesample.

Microwave irradiation can also be used for cell lysing. Notably,microwave cell lysing appears to be related to thermal cell lysing. Inparticular, it has been found that the cell lysing accomplished bymicrowave irradiation can be attributed primarily to thermal effects.See Hiroshi Fujikawa, “Kinetics of Escherichia coli Destruction byMicrowave Irradiation,” Applied and Environmental Microbiology, March,1992, p. 920-24. Thus, microwave irradiation stands as a particularlyconvenient method for heating samples to the extent required for celllysing. In particular, samples can typically be heated for cell lysingmore quickly using microwave irradiation than by conventional heating.This allows greater speed and efficiency in the cell lysing process.Additionally, the microwave cell lysing process is typically easier tocontrol, because the microwave radiation may be easily turned on or offas required. Thus, the possibility of excessive evaporation of thesample is reduced.

However, the benefits of microwave cell lysis are more difficult toapply to small samples, i.e., samples less than one milliliter.Conventional microwave ovens apply microwave radiation at a frequency of2.45 GHz. This frequency is used because of FCC regulations and becausehigh power sources at this frequency are readily available. However, theheating of small samples at this frequency is not very efficient becausethe dimensions of the sample are small compared to the wavelength of themicrowave radiation. This is a significant difficulty because in manycases, particularly when amplification techniques are to be used, onlysmall samples are available.

Accordingly, there is a need to provide more efficient microwave celllysis of small samples.

SUMMARY OF THE INVENTION

In a first principal aspect, the present invention provides a system forheating a sample. The system includes a microwave heating chamber havinga wave-guide cavity with an input port and an output port and means forsupporting the sample in the wave-guide cavity. A microwave sourceproducing microwave radiation at a source power level at a sourcefrequency is coupled to the input port so as to supply microwaveradiation to the input port at an input power level. The sourcefrequency is between 18 and 26 GHz. The microwave exits the output portat an output power level.

In a second principle aspect, the present invention provides a methodfor heating a sample. The sample is placed in a wave-guide cavity havingan input port and an output port. Microwave radiation is applied to theinput port of the wave-guide cavity at an input power level at apredetermined frequency to heat the sample at a predeterminedtemperature for a predetermined time. The predetermined frequency isbetween 18 and 26 GHz. The microwave radiation exits said output port atan output power level.

In a third principal aspect, the present invention provides a method formicrowave cell lysis. The sample, which includes cells, is placed in awave-guide cavity having an input port and an output port. Microwaveradiation is applied to the input port of the wave-guide cavity at aninput power level at a predetermined frequency for a predeterminedperiod of time, the predetermined period of time being sufficient forlysis of said cells. The predetermined frequency is between 18 and 26GHz. The microwave radiation exits said output port at an output powerlevel.

By using microwave radiation with a frequency in the range of 18 to 26GHz the heating of small samples, and, thus, cell lysing, is much moreefficient than when the conventional microwave frequency of 2.45 GHz isused.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of a microwave heating assembly inaccordance with a preferred embodiment of the present invention.

FIG. 2 is a schematic representation of a microwave heating system,which includes the microwave heating assembly of FIG. 1, in accordancewith a preferred embodiment of the present invention.

FIG. 3 is a plot of the measured power loss versus frequency for asample of deionized water placed in the microwave heating system of FIG.2.

FIG. 4 is a plot comparing the temperature rise in a sample of watercaused by microwave heating, using the microwave heating system of FIG.2, with the temperature rise in the sample of water caused by heatingwith a thermal block.

FIG. 5 is a plot of the temperature rise in a sample of E. coli causedby microwave heating, using the microwave heating system of FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1, a microwave heating assembly 8 includes amicrowave heating chamber 10 that defines a wave-guide cavity 12 havingan input port 14 and an output port 16. A sample holder 18 is fittedinto a hole 20 formed into chamber 10. A sample 24 is contained in avial 26, which is supported in sample holder 18 such that vial 26extends into wave-guide cavity 12. Vial 26 is preferably made out of amaterial, such as polypropylene, that is substantially transparent tomicrowaves. In this way, microwave radiation introduced at input port 14may be partially absorbed by sample 24, so as to heat sample 24, andthen exit at output port 16. Preferably, a cap 28 is tightly fitted intothe open top of vial 26 to prevent sample 24 from spilling, such as whensample 24 is heated to boiling, Cap 28 preferably includes a small hole29, through which a thermocouple or other probe may be inserted to reachsample 24.

Shown in FIG. 2 is a microwave heating system 30, which is particularlysuited for heating small samples and for performing microwave cell lysisin small biological samples. System 30 includes microwave heatingassembly 8 and further includes a microwave source 32, such as a solidstate source, that can produce microwave radiation having a sourcefrequency between 18 and 26 GHz. As discussed below, this range offrequencies has been found to be particularly efficient for heatingsmall biological samples. Preferably, microwave source 32 is adjustableso as to allow the source frequency of the microwave radiation producedto be adjusted over the full range of 18 to 26 GHz. Preferably, thesource power level of the microwave radiation produced by microwavesource 32 is also adjustable. Most preferably, microwave source 32 iscomputer-controllable, so as to allow the source frequency and thesource power level to be adjusted by a computer 34. A suitable suchcomputer-controllable microwave source is model HP8340A sold byHewlett-Packard Co.

The output of microwave source 32 may be coupled to an amplifier 35 toprovide a desired gain. In the preferred embodiment, amplifier 35 is atraveling wave tube amplifier, such as model 8001H sold by HughesElectronics Corp. Other types of amplifiers could also be used, however.For example, amplifier 35 may have an adjustable gain that may becontrolled by computer 34.

The output of amplifier 35 is connected to a coupler 36. Coupler 36directs most of the microwave radiation to input port 14, but coupler 36also directs a portion of the microwave radiation to an input powermeasuring instrument 37. Input power measuring instrument 37 can be aspectrum analyzer, power meter, or other device that measures the powerlevel of microwave radiation. Preferably, input power measuringinstrument 37 is a spectrum analyzer that can be interfaced withcomputer 34. A suitable such spectrum analyzer is model HP8563E sold byHewlett-Packard Corp. In this way, computer 34 can monitor the inputpower level, i.e., the power level of the microwave radiation enteringwave-guide cavity 12 at input port 14. Typically, an input power levelon the order of 30 dBm (1 Watt) is suitable for cell lysis whenfrequencies in the range of 18 to 26 GHz are used.

Preferably, the microwave radiation exiting from output port 16 ismeasured by an output power measuring instrument 38, which may bespectrum analyzer, power meter, or other device that can measure thepower level of microwave radiation. Output power measuring instrument 38is preferably a spectrum analyzer that can interface with computer 34,so that computer 34 can monitor the output power level, i.e., the powerlevel of the microwave radiation exiting from output port 16.

Typically, the connections to and from microwave source 32, amplifier35, coupler 36, input power measuring instrument 37, and output powermeasuring instrument 38 will be coaxial connectors having an impedanceof 50 ohms. Accordingly, coaxial to wave-guide adapters (not shown) areconnected to input port 14 and output port 16 to couple the microwaveradiation to wave-guide channel 12. Such coaxial to wave-guide adaptersare commercially available.

Preferably, a thermocouple 40 is inserted through hole 29 in cap 28 andplaced in sample 24 to measure the temperature of sample 24.Thermocouple 40 is connected to a thermocouple reader 42, which measuresthe voltage from thermocouple 40 in comparison with either an internalor external reference to determine the sample temperature. Preferably,thermocouple reader 42 is interfaced with computer 34, so that computer34 can monitor the sample temperature. Although thermocouples areparticularly convenient, other temperature sensors, such as thermistors,or resonant tunneling diodes, could also be used.

System 30, as described above, is designed to be able to provideaccurate temperature control for cell lysis and also to have theflexibility of being able to operate efficiently with a variety ofdifferent types of samples. In using system 30, it is preferable todetermine, by means of input power measuring instrument 37 and outputpower measuring instrument 38, the power loss intrinsic to chamber 10,i.e., with no sample present, over the range of available sourcefrequencies. Then, when sample 24 is added, thee power loss can bemeasured again to determine the absorptance of sample 24. Based on thisabsorptance, computer 34 can then set the source power level ofmicrowave source 32 and/or the gain of amplifier 35 so that the inputpower level will be optimal for cell lysis.

Additionally, computer 34 can monitor the cell lysis process bymeasuring the sample temperature, as described above. Because microwavecell lysis appears to be correlated with heating, the cell lysis processwill typically be controlled by controlling the sample temperature, theduration of a given sample temperature, and the temperature ramp rate.For example, a cell lysis operation may require that the sample bemaintained at a particular temperature, such as 100° C. for a particularperiod of time. By monitoring the sample temperature, and by controllingthe source power level of microwave source 32 and/or the gain ofamplifier 35, computer 34 can control the temperature ramp rate and canmaintain the sample temperature at a predetermined level for apredetermined time, for optimal cell lysis.

It has been found that by using high frequency microwave radiation, theheating of small samples, and, thus, cell lysis in small samples, ismuch more efficient than heating by the 2.45 GHz of conventionalmicrowave ovens. This is believed to result from the shorter wavelengthof the high frequency microwaves being more similar to the dimensions ofthe sample. Additionally, most samples of biological materials arecomposed mostly of water. It is known that pure water has a broad dipoleresonance at a frequency in the vicinity of 21 GHz, depending on thephase, temperature, and the presence of impurities. Thus, the use ofmicrowave radiation in the frequency range of 18 to 26 GHz will beparticularly efficient at heating because of this resonant absorption.

In fact, measurements of the power loss in water, using microwaveheating system 30, demonstrate that the absorptance of microwaveradiation is beneficially high in the frequency range of 18 to 26 GHz,as shown in FIG. 3. Additionally, microwave radiation in this frequencyrange is useful for heating small samples because of the shortwavelengths, relative to the 2.45 GHz used in conventional microwaveovens.

In particular, using microwave heating system 30, it has been found thatwhen microwave radiation in the frequency range of 20 to 22 GHz is usedat an input power level of approximately 30 dBm (1 Watt), a 25microliter sample of deionized water can be heated to its boiling pointin only about 20 seconds, as shown in FIG. 4. In contrast, it was foundthat the same amount of sample could not be heated in a conventionalmicrowave oven operating at 2.45 GHz and a power level of over 600Watts.

Further, as shown in FIG. 4, even after 110 seconds of heating the 25microliter sample using a thermal block, namely a conventional hotplateat a temperature of 120° C., the sample temperature still did not reach100° C.

The results for deionized water have been found also to apply tobiological samples, indicating that the technique is useful formicrowave cell lysis. In particular, as shown in FIG. 5, when usingmicrowave radiation with a frequency of 22 GHz and a power level of 29.7dBm, a 25 microliter sample of E. coli was also able to be heated fromroom temperature to 100° C. in about 20 seconds. FIG. 5 shows two plots:one in which the 100° C. temperature was maintained for 10 seconds andanother plot in which the temperature was maintained for 30 seconds.

With the importance of the frequency of the microwave radiation, it maybe desirable to measure the absorptance of a sample or of a run ofsamples, in order to determine the optimal frequency for cell lysis.This process may be done automatically by computer 34 controlling thesource frequency of microwave source 32. In this way, system: 30 maytake full advantage of the enhancement in cell lysis efficiency that isafforded by high frequency microwave radiation, relative to 2.45 GHzradiation.

In addition to the resonance at approximately 21 GHz, other waterresonances exist at even higher microwave frequencies. For example,water vapor also has resonances at approximately 190 GHz and atapproximately 310 GHz. However, taking advantage of these higherfrequency resonances is more difficult for at least two reasons. First,it is difficult and costly to achieve power levels of even 1 Watt atthese higher microwave frequencies. Second, because the dimensions of awave-guide cavity are inversely proportionally to the frequency, thesehigher frequency resonances would require very small wave-guidedimensions. Such small dimensions would make the construction of athree-dimensional wave-guide cavity, such as provided in chamber 10,more difficult. Additionally, such small wave-guide cavities would notbe able to accommodate conventional sample vials, as does chamber 10 ofthe present invention. It may be possible, however, to constructwave-guide cavities having the required dimensions using moresophisticated techniques. For example, a two-dimensional wave-guidemight be fabricated in an appropriate substrate material. Accordingly,the frequency range of 18 to 26 GHz is particularly beneficial in beingable to take advantage of a microwave water resonance using a wave-guidethat is easy to construct and using microwave power sources andamplifiers that are readily available.

Although an exemplary embodiment has been illustrated and described, itis to be understood that changes and modifications may be made to theinvention without departing from the spirit and scope of the invention,as defined by the following claims.

We claim:
 1. A method for microwave assisted cell lysis, said methodcomprising the steps of: providing a sample, said sample including atleast a plurality of cells, the volume of said sample comprising up toabout 1 mL; providing microwave radiation comprising at least one of apredetermined frequency, a predetermined wavelength and a predeterminedintensity; placing said sample a wave-guide cavity, said wave-guidecavity having an input port and an output port the dimensions of saidwave-guide cavity suitably adapted for effective transmission ofmicrowave radiation in the range of about 18 to about 26 GHz, andapplying to said input of said wave-guide cavity said microwaveradiation at an input power level of said predetermined frequency for apredetermined period of time, said predetermined frequency in the rangeof about 18 to about 26 GHz, said microwave radiation exiting saidoutput port at an output power level, said predetermined period of timebeing sufficient for lysis of at least a portion of said cells.
 2. Themethod of claim 1, further comprising the step of measuring thetemperature of said sample substantially during exposure to saidmicrowave radiation.
 3. The method of claim 2, wherein the sampletemperature is measured with a thermocouple.
 4. The method claim 1,further comprising the step of measuring at least one of said inputpower level and said output power level.
 5. The method of claim 4,further comprising the step of adjusting at least one of saidpredetermined frequency and said input power level as a function of saidoutput power level.
 6. The method of claim 1, wherein said sample iseffectively contained in a vial and at least a portion of said vialremains substantially outside said wave-guide cavity.
 7. The method ofclaim 1, further comprising the steps of: determining the intrinsicpower loss of said cavity; and determining the absorptance of saidsample.
 8. The method of claim 1, further comprising the steps of:measuring said input power level, said output power level and thetemperature of said sample; and adjusting at least one of said inputpower level and said predetermined frequency as a function of at leastone of said input power level, said output power level and thetemperature of said sample.
 9. The method of claim 8, wherein said stepof adjusting is performed substantially automatically by a processor.